1. Field of Invention
The present invention generally relates to the enhancement of immunohistochemical staining of fixed tissue samples. More particularly, an embodiment of the invention relates to the enhancement of immunohistochemical staining of embedded formalin-fixed tissue samples using a single composition in a single step.
2. Description of Related Art
Tissue sections obtained from clinical or animal experimentation frequently have been fixed, embedded and stored in a form suitable for later examination by microscopy. Traditional fixation methods frequently have employed aldehyde fixatives, which fix the tissue by causing cross-linking reactions within and between tissue proteins. Cross-links tend to preserve tissue morphology and integrity, harden the tissue for slicing, and inhibit microbial attack. After tissue samples have been fixed, they are typically embedded in an embedding medium so that the samples may be cut into thin sections. Paraffin is the most common embedding medium, although acrylamide and celloidin may also be used.
Aldehyde fixation tends to cause substantial changes to the structure of the tissue sample. These changes often tend to cause the antigens that may be present in the tissue samples to lose their reactivity toward antibodies that target such antigens. One effect of formalin fixation is to substantially lock the three dimensional shape of protein molecules within the tissue samples. Because of the recent development of new immunohistochemical reagents, immunohistochemical analyses may now be performed that were impossible to perform at the time many tissues were originally stored. Therefore, a number of procedures have been developed which could reverse some of the changes produced by aldehyde fixation, and enhance the immunohistochemical staining properties of the tissue sample.
One method for improving the staining abilities of tissue samples which have been fixed in formalin and embedded in acrylamide gel relates to treatment of acrylamide gel embedded tissue in 1.0% 2-mercaptoethanol for 15 minutes, followed by rinsing with phosphate buffered saline. This treatment allowed the tissue samples to be stained by a number of staining reagents. A method for restoring the imnunohistochemical staining properties of tissue samples is described in U.S. Pat. No. 5,244,787 to Key et al. This method involved removing the embedding medium in a pretreatment step. For Paraffin-embedded tissue samples, this pretreatment was accomplished by e clearing the tissue samples in xylenes and rehydrating the samples. After the embedding medium has been removed, the sample may be heated in either de-ionized water, an aqueous solution of a zinc salt, or an aqueous solution of a lead salt. The tissue samples were reported to show improved immunohistochemical staining properties when heated in a microwave oven. Improvement was reportedly seen when the solution was heated to its boiling temperature. In general, microwave heating appears to have been found by Key et al to give better results than conventional heating. Solutions containing zinc or lead salts apparently gave significantly better results than de-ionized water.
Another method for restoring the immunohistochemical staining properties of tissue samples is described in U.S. Pat. No. 5,578,452 by Shi et al. In this method the formalin-fixed embedded tissues were treated with a solution of an aldehyde releasing reagent. The aldehyde releasing reagent may release aldehyde from the tissue sample by reacting with the aldehyde in a substantially irreversible manner to form a non-aldehyde derivative.
A number of investigators have investigated the importance of several reaction conditions with respect to enhancement of immunohistochemical staining ability. In general it has been found that the pH of the solution and temperature tend to have the most effect on the staining ability of the tissue samples. In general, with some limitations, the higher the temperature during the enhancement of the tissue samples, the better the staining enhancement tends to be. The effect of pH tends to be dependent on the type of antibody being used in the staining process and may be optimized for the antibodies to be used during the staining procedure.
The above mentioned methods inadequately address, among other things, the restoration of paraffin embedded tissue samples in a single reaction step. The procedures described above are usually performed on deparaffinized samples. Typically, the paraffin embedding medium is removed from the samples by successive immersion through a series of xylenes. Following removal of the paraffin embedding medium, the tissue must then be rehydrated by treatment with a series of ethanol-water solutions ranging typically from 100% ethanol to 90% ethanol. Finally, after the sample is rehydrated, the sample must be treated with a solution to reverse the effects of formalin fixation a step known as unmasking. It is therefore desirable that a single solution be provided that allows the steps of deparaffination (or de-embedding), rehydration, and unmasking of embedded tissue samples to be combined.
An embodiment of the invention relates to a liquid composition for enhancing the immunohistochemical staining ability of tissue samples. The composition preferably includes an aqueous solution of a removing agent and a tissue activating agent. The composition preferably substantially simultaneously: (i) removes the embedding medium from the tissue; (ii) improves immunohistochemical staining of the tissue in comparison to tissue that has not been contacted with the composition; and (iii) substantially hydrates the tissue. The removing agent is adapted to substantially remove an embedding medium from a tissue sample. The removing agent is preferably an emulsifier, more preferably a surfactant. The removing agent preferably includes one or more of an amphoteric, anionic, cationic, or nonionic surfactant. The tissue activating agent is adapted to alter the morphology of a component of the tissue sample. The tissue activating agent preferably includes a buffering agent or a metal salt. The pH of the composition is preferably adjusted to lie between 5 to 10 by addition of an acid or base.
In another embodiment the composition preferably includes an aqueous solution of SIMPLE GREEN. Simple Green has been described in U.S. Pat. No. 5,856,289 as: by weight about 5.8% ethylene glycol monobutyl ether, about 3.75% nonylphenol ethoxyate, about 1.5% tetrapotassium pyrophosphate and about 88.95% water. SIMPLE GREEN is a non-toxic, biodegradable, environmentally safe detergent concentrate which may provide a mixture of emulsifiers. The composition may include a buffering agent. The pH of the solution is preferably adjusted to lie between 5 and 10 by addition of an acid or a base. The composition preferably substantially simultaneously: (i) removes the embedding medium from the tissue; (ii) improves immunohistochemical staining of the tissue in comparison to tissue that has not been contacted with the composition; (iii) substantially hydrates the tissue.
In another embodiment of the composition the removing agent includes aqueous solutions of at least one of the following emulsifiers, including detergents and surfactants: Igepal-630 (sigma #3021); Tween 20 (sigma #P7949); Brij 35 (sigma #P 1254); Brij 90 (sigma #P 1254); Triton X-100 (sigma #T9284); CD TAB (sigma #C5335); and Tween 80 (sigma #P8074). Reference to xe2x80x9csigmaxe2x80x9dis reference to (copyright)1999 Sigmaxe2x80x94Aldrich catalog entitled: xe2x80x9cBiochemicals and Reagents for Life Science Researchxe2x80x9d, which is incorporated herein by reference.
The composition is preferably used to enhance the immunohistochemical staining of the tissue sample. In a preferred method the tissue samples are cut into sections of less than 5 microns. The tissue samples may be mounted on a positively charged slide. The sections are preferably dried at about 58xc2x0 C. for one hour. After this time the samples are preferably submersed within the composition. The tissue samples are preferably heated to a temperature of about 120xc2x0 C. at a pressure of about 10 p.s.i. for about 10-15 minutes. After heating the tissue samples are preferably placed in a container containing unused composition at a temperature of at least 90xc2x0 C. for up to about 5 minutes. The tissue sample may be washed with an aqueous solution of a buffering agent before staining. An advantage of the present invention relates to the use of a single composition, in a single step, to enhance the immunohistochemical staining ability of tissue samples.
Another advantage of the present invention is that the sample may be used on Paraffin-embedded tissues without removing the paraffin embedding medium prior to treatment. Yet another advantage of the present invention is to provide a non-toxic, biodegradable composition for pretreatment of slides. Yet another advantage of the present invention is that the composition preferably substantially simultaneously: (i) removes the embedding medium from the tissue; (ii) improves immunohistochemical staining of the tissue in comparison to tissue that has not been contacted with the composition; and (iii) substantially hydrates the tissue.
Tissue sections obtained from clinical or animal experimentation frequently are fixed, embedded and stored in a form suitable for later examination by light microscopy. Traditional fixation methods frequently have employed aldehyde fixatives, especially formaldehyde, which preserves the integrity of the tissue samples as well as protects the sample from microbial attack. Tissue antigens tend to be masked, that is, the antigens within the tissue are no longer reactive toward antibodies. When aldehyde based fixatives are used, this masking of the antigens is thought to be due to the reaction of the aldehyde with the tissue proteins. During the fixation process, the aldehyde presumably fixes the tissue by causing cross-linking reactions within and between tissue proteins, as well as causing other unknown changes to the tissue structure. These cross-links within the tissue proteins tend to alter the three dimensional shape of the protein, preventing access of antibodies to the antigens. After tissue samples have been fixed, they are typically embedded in an embedding medium, such as paraffin or celloidin, so that the samples may be cut into thin sections. The embedding process is preferably accomplished by soaking the tissue samples within the embedding medium such that the tissue samples are substantially surrounded by the embedding medium. In many cases the embedding medium may also soak into the interior of the tissue samples. The embedding medium may prevent the tissue samples from being stained during an immunohistochemical staining procedure.
Recently, new techniques in immunohistochemical staining of tissue samples have been developed. In general tissue samples are studied for the presence of different types of cells. The specific type of cells being studied may be stained, in the presence of other cells, by the application of immunohistochemical staining techniques. During an immunohistochemical staining process the tissue sample may be reacted with an antibody which specifically binds with the type of cells being studied, and no other type of cells. The bound cell-antibody complex may now be stained, without staining any of the other cells, to allow the stained cells to be easily distinguished from the other cells in the tissue sample. These techniques typically require the antigens to be unmasked before use. Because of the wide spread use of formaldehyde as a fixation chemical or a constituent thereof, it is desirable to provide a procedure whereby the antigenic activity of these tissue samples may be restored. Such a procedure may also take into account the embedding medium of the tissue samples. Formalin-fixed tissue samples are commonly embedded in paraffin before use. Removal of a portion of the embedding medium is thought to be necessary before immunohistochemical staining may be accomplished.
The enhancement of immunohistochemical staining of embedded, fixed tissue samples (typically formalin-fixed tissue samples) may be achieved by treating the tissue samples with an appropriate liquid composition. The composition preferably improves the staining of the tissue samples by accomplishing three effects. These effects may be accomplished substantially simultaneously. First, the composition may substantially remove the embedding medium from the tissue sample. Removal of the embedding medium allows the penetration of the immunohistochemical stains into at least a portion of the tissue samples preserved by the fixation process. Finally, the composition preferably rehydrates the tissue sample. During the process of fixation most of the water is removed from the tissue sample, rehydration of the tissue sample may allow the tissue to change its morphological structure and/or attain its original morphological structure.
An embodiment of a liquid composition for enhancing the immunohistochemical staining ability of embedded tissue samples includes a removing agent and a tissue activating agent. The composition may be a solution of the removing agent and the tissue activating agent in a solvent. The composition preferably is an aqueous solution of the removing agent and the tissue activating agent. The removing agent is preferably adapted to substantially remove an embedding medium from the tissue sample. During the process of embedding, the tissue becomes substantially surrounded with the embedding medium such that the embedding medium may inhibit the tissue from reacting with antibodies. To enhance the immunohistochemical staining of the tissue samples, at least a portion of the embedding medium is preferably removed from the tissue sample. To achieve the best results it is preferred that substantially all of the embedding medium is removed.
In some cases the embedding medium may be substantially water soluble. In this case water may act as the removing agent as well as a solvent for the tissue activating agent. If the embedding medium is a non-polar medium, such as paraffin, and the composition is an aqueous solution, the removing agent may induce the formation of an emulsion of the embedding medium within the composition. The term xe2x80x9cemulsionxe2x80x9d within the context of this application is taken to mean a stable mixture of two or more immiscible liquids held in suspension. The mixture may be held in suspension by one or more surfactants. An elevated temperature may be necessary to maintain such suspension.
The removing agent is preferably a surfactant. A surfactant is any compound that reduces the interfacial tension between two liquids or between a liquid and a solid. In general surfactants allow a solid or liquid, normally immiscible with a solvent, to become finely suspended with the solvent. Surfactants are typically divided into four classes: amphoteric, anionic, cationic, and non-ionic. The removing agent may be taken from any of these classes of surfactants. Preferably, the removing agent may be comprised of a mixture of compatible surfactants taken from one or more of these classes.
Nonionic surfactants include molecules that contain a substantially polar functional group attached to a substantially non-polar group.
The tissue activating agent is preferably adapted to interact with the tissue sample such that the morphology of the components of the tissue are altered. In general the tissue sample contains a number of biological components, including proteins and nucleic acids. Each of these components have a specific three dimensional structure related to the composition of the component. During the fixation, the tissue sample may be treated with an aqueous solution of formaldehyde. The formaldehyde reacts with the components to alter the three dimensional shape of these components, i.e. alter the morphology. These alterations tend to make the tissue samples substantially unreactive toward various immunohistochemical staining protocols. The tissue activating agent is preferably adapted to further alter the morphology of the tissue samples, such that the tissue samples are more reactive toward immunohistochemical reagents. While the tissue activating reagent restores some of the reactivity of the tissue samples, it may not be necessary that the tissue be restored to its original morphology to increase the reactivity of the samples toward immunohistochemical stains.
A number of metal salts may be used as a tissue activating agent. Salts useful in the composition include, but are not limited to: aluminum chloride, sodium chloride, sodium fluoride, iron chloride, zinc sulfate, and lead thiocyanate. In general aqueous solutions containing these and other metal salts improve the immunohistochemical staining ability of tissue samples to a greater extent than de-ionized water. It is believed that these salts may exert an effect on the morphology, i.e. three dimensional shape, of the protein components. The interaction of these salts with the protein molecule may aid in allowing the protein to attain a substantial part of its original three dimensional shape.
Chelators may be used as tissue activating agents. These chelators include: EDTA (sigma #E9884); EGTA (sigma #E4378); CDTA (sigma #D0922); EDADP-X (sigma #E1254); EDADP-B (sigma #E2004); EDDRA (sigma #E4135); DFA: (sigma #D9533); PPi (sigma #P8010); EDTP-B and EDTP-X. Of these, the preferred chelators are EDDHA, EDTA and EGTA. The concentration of these chelators as tissue activating agents is preferably in the range of 1 to 2 millimolar.
Buffering agents may also be used as a tissue activating agent. Buffering agents useful in the composition include, but are not limited to: citric acid, tartrate salts, phthalate salts, borate salts, tris(hydroxymethyl)aminomethane (Tris-HCI), EDTA and phosphate salts. In general the choice of the buffer is dependent on the desired pH range of the composition. By choosing the appropriate buffer the pH of the composition may be altered to values ranging from about 1 to 11. Preferably the pH of the composition is maintained between about 4 and 10 more preferably between about 5 and 8. In general buffering agents modify the morphology of the proteins. Aqueous solutions containing these and other buffering agents improve the immunohistochemical staining ability of tissue samples to a greater extent than de-ionized water. The staining enhancement of tissue samples varies with the pH of the solution. In general, most tissue samples show the most enhancement at pH values between 5 and 8. Some tissue samples, however, do not show significant improvements at these pH levels.
The preferred buffers for a high pH (about 10) are ethanolamine (sigma #E9508), diethanolamine (DEA) (sigma #D068 1) and DEA-tris.
The composition may optionally include a dye or other substances that do not significantly contribute to the function of the composition. These substances are typically added to allow easy identification of the composition or to improve the odor of the composition.
According to a preferred embodiment, the composition is an aqueous solution which includes (a) a removing agent and (b) a tissue activating agent. The composition may optionally include a dye.
In a preferred embodiment, the composition is an aqueous solution which includes (a) up to about 25 percent by volume of SIMPLE GREEN and (b) up to about 10 percent by weight of citric acid and alkaline citrate salts. In a more preferred embodiment the composition is an aqueous solution which includes (a) about 1.85 percent by volume of SIMPLE GREEN, (b) about 0.3 percent by weight of trisodium citrate [dehydrate] dihydrate; and (c) sufficient concentrated hydrochloric acid to obtain a pH of about 5.96 to 6.04.
The composition, as described in previous embodiments, may be used to enhance the immunohistochemical staining ability of formalin-fixed embedded tissue samples. Tissue samples are preferably prepared by soaking the tissue in a buffered formalin or other fixative solution. After soaking for the appropriate time, the tissue sample is preferably dehydrated in ethanol, cleared using xylenes and embedded into paraffin blocks. The embedded tissue is preferably cut into sections up to about 5 micron sections; more preferably into about 3 micron sections. The tissue sample may be mounted onto a positively charged slide. The tissue sample is preferably mounted onto a poly-L-lysine coated slide. Poly-L-lysine is a positively charged, high molecular weight polymer of the amino acid lysine which, when coated onto a microscope slide, tends to act as a tissue adhesive bonding the tissue to the slide. The use of a tissue adhesive may be necessary to prevent the detachment of the tissue sample from the slide. After the samples have been mounted they may be dried for at least one hour at a temperature of about 58xc2x0 C.
The mounted samples may then be contacted with a composition, prepared according to the previously described embodiments, and heated to a temperature of at least about 80xc2x0 C. for a time period of at least about 10 minutes. The samples may be contacted with the composition by submersing a portion of the tissue sample within the composition. The samples are preferably substantially submerged within the composition. Heating may be accomplished by a number of heating devices including, but not limited to: autoclaves, pressure cookers, water baths, microwave ovens, and steam heating. In general it is preferred that the slides are heated to a temperature of at least 80xc2x0 C. for a time period of at least 50 minutes; more preferably the slides are heated at least about 100xc2x0 C. for a time period of at least about 30 minutes; more preferably still the slides are heated at a temperature of at least about 110xc2x0 C. for a period of at least about 20 minutes. The slides may be heated at atmospheric pressure. When a pressure cooker is used as the heating source the slides are preferably heated to a temperature up to about 120xc2x0 C. and a pressure of up to about 2 atmospheres.
The heating is preferably sustained for a time such that the samples also become substantially hydrated. During the embedding process the water in the tissue samples is typically removed by washing the tissue samples in an alcohol. To perform an immunohistochemical staining procedure upon the tissue samples it is preferred that the tissue is substantially saturated in water. These hydrated tissue samples typically exhibit improved immunohistochemical staining over unhydrated tissue samples.
After the slides have been heated in the composition they may be washed with an appropriate buffer solution to remove composition remaining on the slide. Alternatively, after the slides have been heated in the composition, they may be removed and washed with a second composition prepared according to the previously described embodiments. The second composition may include the same components as the initial composition. The second composition may be at least at room temperature. Preferably, the second composition is heated to a temperature of at least about 90xc2x0 C. before the slides are contacted with the second composition. After contacting the slides with the second composition for about 5 minutes the slides may be additionally washed with an appropriate buffer solution to remove any of the composition remaining on the slide. The tissue samples may be stained at this point according to standard immunohistochemical staining protocols.